Polyol additives modulate the in vitro stability and activity of recombinant human phenylalanine hydroxylase

Area of Science:

• Biochemistry
• Enzymology
• Metabolic Disorders

Background:

• Phenylketonuria (PKU) is a common amino acid metabolism disorder caused by deficient phenylalanine hydroxylase (hPAH) activity.
• Dietary management prevents neurodevelopmental issues, but new therapies like enzyme replacement are sought.
• In vitro instability of hPAH hinders its use in enzyme replacement therapy.

Purpose of the Study:

• To evaluate the efficacy of polyol compounds in stabilizing purified hPAH in vitro.
• To assess the impact of glycerol, mannitol, and PEG-6000 on hPAH stability under different storage conditions.

Main Methods:

• Purified recombinant hPAH was stored with polyol stabilizers (glycerol, mannitol, PEG-6000) at 4°C and -20°C for one month.
• Enzyme stability was assessed by monitoring protein content, degradation products, specific activity, oligomeric profile, and conformational changes.
• Thermodynamic parameters (DeltaG(0)) and quenching parameters were determined.

Main Results:

• 50% glycerol or 10% mannitol at -20°C effectively protected hPAH from activity loss.
• Stabilizing agents prevented degradation and maintained the enzyme's oligomeric profile.
• Conformational changes, indicated by thermodynamic and quenching parameters, correlated with enhanced catalytic efficiency.

Conclusions:

• Glycerol and mannitol show promise as effective stabilizers for recombinant hPAH in vitro.
• Optimized stabilization could overcome hPAH instability, supporting its potential for PKU enzyme replacement therapy.
• Further research into the conformational effects of stabilizers may enhance enzyme performance.